MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

miR‐193a/b‐3p relieves hepatic fibrosis and restrains proliferation and activation of hepatic stellate cells

MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR‐193a/b‐3p in concanavalin A (ConA)‐induced liver fibrosis in mice was evaluated. According to the results, the expression of miR‐193a/b‐3p was down‐regulated in liver tissues after exposure to ConA. Lentivirus‐mediated overexpression of miR‐193a/b‐3p reduced ConA‐induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA‐induced liver fibrosis was restrained by the up‐regulation of miR‐193a/b‐3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor‐β1 (TGF‐β1) and activin A in liver tissues. Furthermore, miR‐193a/b‐3p mimics suppressed the proliferation of human HSCs LX‐2 via inducing the apoptosis of LX‐2 cells and lowering the levels of cell cycle‐related proteins Cyclin D1, Cyclin E1, p‐Rb and CAPRIN1. Finally, TGF‐β1 and activin A‐mediated activation of LX‐2 cells was reversed by miR‐193a/b‐3p mimics via repressing COL1A1 and α‐SMA expression, and restraining the activation of TGF‐β/Smad2/3 signalling pathway. CAPRIN1 and TGF‐β2 were demonstrated to be the direct target genes of miR‐193a/b‐3p. We conclude that miR‐193a/b‐3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR‐193a‐3p and miR‐193b‐3p may be new therapeutic targets for liver fibrosis.     

MiR‐34b‐3p represses cell proliferation,cell cycle progression and cell apoptosis in non‐small‐cell lung cancer (NSCLC) by targeting CDK4

Lung cancer is the most common incident cancer, with a high mortality worldwide, and non‐small‐cell lung cancer (NSCLC) accounts for approximately 85% of cases. Numerous studies have shown that the aberrant expression of microRNAs (miRNAs) is associated with the development and progression of cancers. However, the clinical significance and biological roles of most miRNAs in NSCLC remain elusive. In this study, we identified a novel miRNA, miR‐34b‐3p, that suppressed NSCLC cell growth and investigated the underlying mechanism. miR‐34b‐3p was down‐regulated in both NSCLC tumour tissues and lung cancer cell lines (H1299 and A549). The overexpression of miR‐34b‐3p suppressed lung cancer cell (H1299 and A549) growth, including proliferation inhibition, cell cycle arrest and increased apoptosis. Furthermore, luciferase reporter assays confirmed that miR‐34b‐3p could bind to the cyclin‐dependent kinase 4 (CDK4) mRNA 3′‐untranslated region (3′‐UTR) to suppress the expression of CDK4 in NSCLC cells. H1299 and A549 cell proliferation inhibition is mediated by cell cycle arrest and apoptosis with CDK4 interference. Moreover, CDK4 overexpression effectively reversed miR‐34‐3p‐repressed NSCLC cell growth. In conclusion, our findings reveal that miR‐34b‐3p might function as a tumour suppressor in NSCLC by targeting CDK4 and that miR‐34b‐3p may, therefore, serve as a biomarker for the diagnosis and treatment of NSCLC.     

MiR‐29b‐3p promotes chondrocyte apoptosis and facilitates the occurrence and development of osteoarthritis by targeting PGRN

This study was aimed to explore the role of miR‐29b‐3p and PGRN in chondrocyte apoptosis and the initiation and progress of osteoarthritis (OA). Both miR‐29b‐3p and PGRN were up‐regulated in cartilage tissue from patients with OA. Transfection of miR‐29b‐3p mimic into rat primary chondrocytes and SW1353 chondrosarcoma cells significantly suppressed PGRN expression and release, induced apoptosis, inhibited proliferation and scratch wound closure. By contrast, transfection of miR‐29b‐3p inhibitor exhibited the opposite effects. Moreover, the expression and secretion of cartilaginous degeneration‐related molecules were also altered by miR‐29b‐3p. Luciferase reporter gene assay showed rat GRN mRNA is directly targeted and repressed by miR‐29b‐3p. The fact that recombinant PGRN or shPGRN‐mediated PGRN interference abolished miR‐29b‐3p mimic‐induced cell apoptosis and growth inhibition suggested miR‐29b‐3p affect the cellular functions of chondrocyte through regulating PGRN expression. In vivo, joint cavity injection of miR‐29b‐3p antagomir prior to surgical induction of OA significantly suppressed the upregulation of miR‐29b‐3p, whereas further promoted the increased expression of PGRN. Articular chondrocytes apoptosis and cartilage loss in the knee joint of surgically induced OA rats were also ameliorated by the injection of miR‐29b‐3p antagomir, demonstrated by TUNEL and safranin O‐fast green staining. This work showed miR‐29b‐3p facilitates chondrocyte apoptosis and OA by targeting PGRN, and miR‐29b‐3p or PGRN may be the potential target for OA treatments.     

MicroRNA‐130b targets PTEN to induce resistance to cisplatin in lung cancer cells by activating Wnt/β‐catenin pathway

More and more studies indicate the relevance of miRNAs in inducing certain drug resistance. Our study aimed to investigate whether microRNA‐130b‐3p (miR‐130b) mediates the chemoresistance as well as proliferation of lung cancer (LC) cells. MTS assay and apoptosis analysis were conducted to determine cell proliferation and apoptosis, respectively. Binding sites were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT‐PCR and immunoblot, respectively. Mouse xenograft model was used to evaluate the role of miR‐130b in cisplatin resistance in vivo. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR ) versus its parental cell lines, indicated its crucial relevance for LC biology. We identified PTEN as miR‐130b's major target and inversely correlated with miR‐130b expression in LC. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. Suppression of miR‐130b enhanced drug cytotoxicity and reduced proliferation of A549/CR cells both internally and externally. Particularly, miR‐130b mediated Wnt/β‐catenin signalling pathway activities, chemoresistance and proliferation in LC cell, which was partially blocked following knockdown of PTEN. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR) versus its parental cell lines, indicated its crucial relevance for LC biology. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway.     

Up‐regulation of miR‐10b‐3p promotes the progression of hepatocellular carcinoma cells via targeting CMTM5

In this study, we investigated how miR‐10b‐3p regulated the proliferation, migration, invasion in hepatocellular carcinoma (HCC) at both in vitro and in vivo levels. CMTM5 was among the differentially expressed genes (data from TCGA). The expression of miR‐10b‐3p and CMTM5 was detected by qRT‐PCR and Western blot (WB). TargetScan was used to acquire the binding sites. Dual‐luciferase reporter gene assay was used to verify the direct target relationship between miR‐10b‐3p and CMTM5. WB analysis proved that miR‐10b‐3p suppressed CMTM5 expression. Furthermore, proliferation, invasion and migration of HCC cells were measured by MTT assay, colony formation assay, transwell assay and wound‐healing assay, respectively. Kaplan‐Meier plotter valued the overall survival of CMTM5. Finally, xenograft assay was also conducted to verify the effects of miR‐10b‐3p/CMTM5 axis in vivo. Up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were detected in HCC tissues and cell lines. CMTM5 was verified as a target gene of miR‐10b‐3p. The overexpression of CMTM5 contributed to the suppression of the proliferative, migratory and invasive abilities of HCC cells. Moreover, the up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were observed to be associated with worse overall survival. Lastly, we have confirmed the carcinogenesis‐related roles of miR‐10b‐3p and CMTM5 in vivo. We concluded that the up‐regulation of miR‐10b‐3p promoted the progression of HCC cells via targeting CMTM5.     

Effect of the interaction between MiR‐200b‐3p and DNMT3A on cartilage cells of osteoarthritis patients

The aim of this research is to explore the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of osteoarthritis (OA) cartilage cells. Quantitative RT‐PCR was performed to analyse the expression of miR‐200b‐3p, DNMT3A, MMP1, MMP3, MMP9, MMP13 and COL II in normal and OA cartilage tissues. The dual‐luciferase reporter assay and Western blot assay were conducted to confirm the targeting relationship between miR‐200b‐3p and DNMT3A. We also constructed eukaryotic expression vector to overexpress miR‐200b‐3p and DNMT3A. We detected the expression level of MMPs and COL II in stable transfected cartilage cells using RT‐PCR and Western blot. Cell proliferation and apoptosis were evaluated using the MTS, pellet culture and Hoechst 33342 staining method. Finally, we explored the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of OA cartilage cells. The results of RT‐PCR indicated that both miR‐200b‐3p and COL II were down‐regulated in OA cartilage tissues, while the expression of DNMT3A and MMPs was up‐regulated in OA cartilage tissues. The expressions of DNMT3A, MMPs and COL II detected by Western blot showed the same trend of the results of RT‐PCR. The dual‐luciferase reporter assay and Western blot assay confirmed the targeting relationship between miR‐200b‐3p and DNMT3A. In overexpressed miR‐200b‐3p cartilage cells, DNMT3A and MMPs were significantly down‐regulated, COL II was significantly up‐regulated, cell viability was enhanced and apoptosis rate was decreased (P < 0.05). In overexpressed DNM3T cartilage cells, MMPs were significantly up‐regulated, COL II was significantly down‐regulated, cell viability was weakened and apoptosis rate was increased (P < 0.05). MiR‐200b‐3p inhibited the secretion of MMPs, promoted the synthesis of COL II and enhanced the growth and proliferation of OA cartilage cells through inhibiting the expression of DNMT3A.     

miR‐4732‐5p promotes breast cancer progression by targeting TSPAN13

MiR‐4732‐5p was previously found to be dysregulated in nipple discharge of breast cancer. However, the expression and function of miR‐4732‐5p in breast cancer remain largely unknown. Here, the expression of miR‐4732‐5p was detected using quantitative real‐time PCR in breast cancer tissues and cell lines. Cell proliferation, apoptosis, migration and invasion assays were performed to examine the effects of miR‐4732‐5p in breast cancer. In addition, mRNA sequencing, bioinformatics analysis, Western blot and luciferase assays were performed to identify the target of miR‐4732‐5p. Overall, miR‐4732‐5p was significantly down‐regulated in breast cancer tissues, especially in lymph node metastasis (LNM)‐negative tissues, compared with adjacent normal tissues. However, it was more highly expressed in LNM‐positive breast cancer tissues, compared with LNM‐negative ones. Expression of miR‐4732‐5p was positively correlated with lymph node metastasis, larger tumour size, advanced clinical stage, high Ki‐67 levels and poor prognosis. MiR‐4732‐5p promoted cell proliferation, migration and invasion in breast cancer. MiR‐4732‐5p directly targeted the 3′‐UTR of tetraspanin 13 (TSPAN13) and suppressed TSPAN13 expression at the mRNA and protein levels. These results suggested that miR‐4732‐5p may serve as a tumour suppressor in the initiation of breast cancer, but as a tumour promoter in breast cancer progression by targeting TSPAN13.     

Novel role of the clustered miR‐23b‐3p and miR‐27b‐3p in enhanced expression of fibrosis‐associated genes by targeting TGFBR3 in atrial fibroblasts

Atrial fibrillation (AF) is the most common type of arrhythmia in cardiovascular diseases. Atrial fibrosis is an important pathophysiological contributor to AF. This study aimed to investigate the role of the clustered miR‐23b‐3p and miR‐27b‐3p in atrial fibrosis. Human atrial fibroblasts (HAFs) were isolated from atrial appendage tissue of patients with sinus rhythm. A cell model of atrial fibrosis was achieved in Ang‐II‐induced HAFs. Cell proliferation and migration were detected. We found that miR‐23b‐3p and miR‐27b‐3p were markedly increased in atrial appendage tissues of AF patients and in Ang‐II‐treated HAFs. Overexpression of miR‐23b‐3p and miR‐27b‐3p enhanced the expression of collagen, type I, alpha 1 (COL1A1), COL3A1 and ACTA2 in HAFs without significant effects on their proliferation and migration. Luciferase assay showed that miR‐23b‐3p and miR‐27b‐3p targeted two different sites in 3?‐UTR of transforming growth factor (TGF)‐β1 receptor 3 (TGFBR3) respectively. Consistently, TGFBR3 siRNA could increase fibrosis‐related genes expression, along with the Smad1 inactivation and Smad3 activation in HAFs. Additionally, overexpression of TGFBR3 could alleviate the increase of COL1A1, COL3A1 and ACTA2 in HAFs after transfection with miR‐23b‐3p and miR‐27b‐3p respectively. Moreover, Smad3 was activated in HAFs in response to Ang‐II treatment and inactivation of Smad3 attenuated up‐regulation of miR‐23b‐3p and miR‐27b‐3p in Ang‐II‐treated HAFs. Taken together, these results suggest that the clustered miR‐23b‐3p and miR‐27b‐3p consistently promote atrial fibrosis by targeting TGFBR3 to activate Smad3 signalling in HAFs, suggesting that miR‐23b‐3p and miR‐27b‐3p are potential therapeutic targets for atrial fibrosis.     

MiR‐376a suppresses the proliferation and invasion of non‐small‐cell lung cancer by targeting c‐Myc

It has been reported that miR‐376a is involved in the formation and progression of several types of cancer. However, the expression and function of miR‐376a is still unknown in non‐small cell lung carcinomas (NSCLC). In this study, the expression of miR‐376a in NSCLC tissues and cell lines were examined by real‐time PCR, the effects of miR‐376a on cell proliferation, apoptosis and invasion were evaluated in vitro. Luciferase reporter assay was performed to identify the targets of miR‐376a. The results showed that miR‐376a was significantly downregulated in NSCLC tissues and cell lines. Restoration of miR‐376a in NSCLC cell line A549 significantly inhibited cell proliferation, increased cell apoptosis and suppressed cell invasion, compared with control‐transfected A549 cells. Luciferase reporter assay showed that c‐Myc, an oncogene that regulating cell survival, angiogenesis and metastasis, was a direct target of miR‐376a. Over‐expression of miR‐376a decreased the mRNA and protein levels of c‐Myc in A549 cells. In addition, upregulation of c‐Myc inhibited miR‐376a‐induced inhibition of cell proliferation and invasion in A549 cells. Therefore, our results indicate a tumor suppressor role of miR‐376a in NSCLC by targeting c‐Myc. miR‐376a may be a promising therapeutic target for NSCLC.     

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR‐9‐5p/QKI‐5 axis

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT‐PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR‐9‐5p and QKI‐5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK‐8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR‐9‐5p, whereas miR‐9‐5p down‐regulated QKI‐5 expression. Overexpressed MEG3 and QKI‐5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR‐9‐5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up‐regulated expression of QKI‐5 in vivo. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR‐9‐5p and its targeting gene QKI‐5.     

Long noncoding RNA HNF1A‐AS1 regulates proliferation and apoptosis of glioma through activation of the JNK signaling pathway via miR‐363‐3p/MAP2K4

Long noncoding RNAs (lncRNAs) have been proven to exert important functions in the various biological processes of human cancers. It has been reported that lncRNA HNF1 homeobox A antisense RNA 1 (HNF1A‐AS1) was abnormally expressed and played a role in the initiation and development of various human cancers. In this study, we confirmed that the expression level of HNF1A‐AS1 was increased in glioma tissues and cells. Knockdown of HNF1A‐AS1 inhibited cell proliferation and promoted cell apoptosis in glioma. Then, we disclosed the downregulation of miR‐363‐3p in glioma tissues and cell lines. The interaction between HNF1A‐AS1 and miR‐363‐3p was identified in glioma cells. Furthermore, an inverse correlation between HNF1A‐AS1 and miR‐363‐3p was observed in glioma tissues. Afterwards, we recognized that MAP2K4 was a direct target of miR‐363‐3p. The expression of MAP2K4 was negatively correlated with miR‐363‐3p while positively related to HNF1A‐AS1 in glioma tissues. We also found the regulatory effect of HNF1A‐AS1 on the MAP2K4‐dependent JNK signaling pathway. All findings indicated that HNF1A‐AS1 induces the upregulation of MAP2K4 to activate the JNK signaling pathway to promote glioma cell growth by acting as a miR‐363‐3p sponge.     

LINC01303 functions as a competing endogenous RNA to regulate EZH2 expression by sponging miR‐101‐3p in gastric cancer

Long non‐coding RNA (lncRNA) is one of the important regulators of many malignancies. However, the biological function and clinical significance of a large number of lncRNAs in gastric cancer remain unclear. Therefore, we analysed the TCGA data to find that LINC01303 is significantly up‐regulated in gastric cancer tissues. However, the biological function of LINC01303 in GC remains unknown. In our study, we found that the expression of LINC01303 was significantly higher in GC tissues than in adjacent tissues by real‐time quantitative PCR. We can significantly inhibit the malignant proliferation, migration and invasion of GC cells by silencing LINC01303 expression. In addition, LINC01303 knockdown can also inhibit GC growth in vivo. After the bioinformatics analysis, we found that LINC01303 can be used as a miR‐101‐3p sponge to competitively adsorb miR‐101‐3p with EZH2. Therefore, our results indicate that LINC01303 promotes the expression of EZH2 by inhibiting miR‐101‐3p activity and promotes GC progression. In summary, in this study, we demonstrated for the first time that the LINC01303/miR‐101‐3p/EZH2 axis promotes GC progression.     

Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p

Long non‐coding RNAs (lncRNAs) could regulate growth and metastasis of hepatocellular carcinoma (HCC). In this study, we aimed to investigate the mechanism of lncRNA F11‐AS1 in hepatitis B virus (HBV)–related HCC. The relation of lncRNA F11‐AS1 expression in HBV‐related HCC tissues to prognosis was analysed in silico. Stably HBV‐expressing HepG2.2.15 cells were established to explore the regulation of lncRNA F11‐AS1 by HBx protein, as well as to study the effects of overexpressed lncRNA F11‐AS1 on proliferation, migration, invasion and apoptosis in vitro. Subsequently, the underlying interactions and roles of lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis in HBV‐related HCC were investigated. Additionally, the influence of lncRNA F11‐AS1 and miR‐211‐5p on tumour growth and metastasis capacity of HepG2.2.15 cells were studied on tumour‐bearing nude mice. Poor expression of lncRNA F11‐AS1 was correlated with poor prognosis in patients with HBV‐related HCC, and its down‐regulation was caused by the HBx protein. lncRNA F11‐AS1 was proved to up‐regulate the NR1I3 expression by binding to miR‐211‐5p. Overexpression of lncRNA F11‐AS1 reduced the proliferation, migration and invasion, yet induced apoptosis of HepG2.2.15 cells in vitro, which could be abolished by overexpression of miR‐211‐5p. Additionally, either lncRNA F11‐AS1 overexpression or miR‐211‐5p inhibition attenuated the tumour growth and metastasis capacity of HepG2.2.15 cells in vivo. Collectively, lncRNA F11‐AS1 acted as a modulator of miR‐211‐5p to positively regulate the expression of NR1I3, and the lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis participated in HBV‐related HCC progression via interference with the cellular physiology of HCC.     

MicroRNA‐155‐3p promotes glioma progression and temozolomide resistance by targeting Six1

The prognosis of glioma is generally poor and is the cause of primary malignancy in the brain. The role of microRNAs has been implicated in tumour inhibition or activation. In several cancers, the Six1 signalling pathway has been found to be aberrant and also relates to the formation of tumours. We analysed the database for expression profiles and clinical specimens of various grades of glioma to assess microRNA‐155‐3p (miR‐155‐3p) expression. The role of miR‐155‐3p in glioblastoma, cell cycle, proliferation, apoptosis and resistance to temozolomide was assessed in vitro through flow cytometry and cell proliferation assays. Bioinformatics analyses, and assays using luciferase reporter, and immunoblotting revealed that miR‐155‐3p targets Six1 and that the relationship between glioma and healthy brain tissues was significantly inverse. In rescue experiments, overexpressed Six1 revoked the changes in cell cycle distribution, proliferation and resistance to temozolomide estimated by apoptosis induced by overexpressed miR‐155‐3p. MiR‐155‐3p inhibition reduced glioma cell growth and proliferation in the brain of a mouse model and increased the survival of mice with gliomas. Thus, miR‐155‐3p modulates Six1 expression and facilitates the progression of glioblastoma and resistance to temozolomide and may act as a novel diagnostic biomarker and a target for glioma treatment.